Process optimization and antioxidative activity of polyphenols derived from different seaweed species Sargassum Miyabei, Undaria Pinnatifida Suringar, and Sargassum Thunbergii

Abstract The aim of this study was to extract the polyphenols from three major seaweed species such as Sargassum miyabei, Undaria pinnatifida suringar, and Sargassum thunbergii, which are found in the coastal province (Guangdong), a longest coastal line in China. It was found that the Sargassum thunbergii produced more polyphenols (34.99 mg) as compared to Sargassum miyabei (23.26 mg) and Undaria pinnatifida suringar (25.34 mg), respectively. The orthogonal method was used for the extraction of phenolic compounds and extraction condition of each seaweed species was optimized. The antioxidant activity of extracted polyphenols from all three species stated that the polyphenols extracted from Undaria pinnatifida suringar demonstrated the highest antioxidative activity. Furthermore, gas chromatography–mass spectrometry (GC‐MS) was used for qualitative analysis of polyphenols, which revealed that the major components of polyphenols extracted from Undaria pinnatifida suringar were gallic acid and arbutin followed by syringate in Sargassum miyabei and phloretin in Sargassum thunbergii.

for the extraction of polyphenolic compounds and reported that the total phenolic content of these 12 seaweed species varied greatly, i.e., from 2% to 25%. Rajauria et al. (2016) extracted polyphenolic compounds from the Irish seaweed Himanthalia elongate followed by the separation of the crude extract by applying various techniques.
They stated that the phenolic contents varied from 5.25% to 31% as separated by liquid-liquid partition-column chromatographymethanol subfraction and liquid-liquid partition-ethyl acetate fraction, respectively. China and Indonesia are the largest seaweed producers with over 23 million tons of aggregated seaweed production in the world. Each produced more than 10 million tons of seaweed in 2017 (Buschmann et al., 2017). Among them, Guangdong Province is a major hub of seafood production including seaweed cultivation and production. It is the longest coastline (8500 km) covering all Chinese provinces, and approximately a fifth of the seafood production (over 180,000 tons in 2017). However, the efficiency of polyphenolic compound extraction from the major seaweed species of Guangdong Province has not yet been comprehensively studied and compared.
The antioxidative activity of polyphenolic compounds extracted from seaweed has been broadly studied. In this regard, Kajal et al. (2015) used different in vitro systems (DPPH, ABTS, HO radical scavenging activities, H 2 O 2 scavenging ability, and Fe 2+ ion chelating ability) to evaluate the antioxidative activity of three seaweeds (Hypnea musciformis, H. valuentiae, and Jania rubens) collected from the Gulf of Mannar on the southeastern coast of India.
They indicated that H. valuentiae was the best specie among the three seaweeds due to its higher antioxidant activity and could be used as a potential food preservative. Shipeng et al. (2015) applied a more advanced technique, supercritical carbon dioxide extraction, to produce seaweed oil from the Sargassum horneri seaweed and reported that the extracted oil had a significant correlation between the antioxidative activity and polyphenolic content.
The extracted seaweed oil with the highest antioxidative activity (68.38% measured by the DPPH method and 83.51% measured by the ABTS method) also contained the highest total polyphenolic content (0.64 mg/g). However, breakdown studies regarding the composition of each polyphenolic compound in the extracted polyphenolic complex and the relationship between this composition and the antioxidative activity of the polyphenolic complex have rarely been previously conducted.
Among the various seaweeds found along the coastline of Guangdong Province, Sargassum miyabei, Undaria pinnatifida suringar, and Sargassum thunbergii are the most abundantly available irrespective of the season (Shipeng et al., 2015). Although the antioxidative activity of polyphenolic compounds extracted from seaweeds has been proven by many previous studies, there is scant information regarding the antioxidative effects of the polyphenolic compounds extracted from these species, and from this crucial costal region of China. The results from this study will fulfill this knowledge gap.
Furthermore, the seaweed specie with the best efficiency of polyphenolic compounds extraction and the polyphenols contributing the most toward antioxidative activity can be found. Therefore, this study will provide valuable information for the development of regional seaweed production in Guangdong Province, China, from both scientific and industrial points of view.

| Materials
The three seaweed species Sargassum miyabei, Undaria pinnatifida suringar, and Sargassum thunbergii were collected from the ocean region close to the coastal line of Guangdong Province, China. All chemicals were purchased from Sigma-Aldrich Corporation.

| Processing optimization of polyphenol complexes extracted from seaweeds
Extraction time, extraction temperature, concentration of ethanol, and the ratio of seaweed to extraction liquid were considered as four key factors for optimization. Each factor was tested at three levels ( Table 1). The experiments were carried out according to the orthogonal design method. Five grams of each dried seaweed was mixed with ethanol in designed ratios (1:8, 1:9, and 1:10, Table 1). Three different concentrations (70%, 80%, and 90%) of ethanol were set for the trials (Table 1). The mixture of dried seaweed powder and ethanol was placed in a water bath and sonicated for the designed time (3 h, 4 h, and 5 h) and temperature (50°C, 60°C, and 70°C) ( Table 1), then filtered with suction on No. 617 paper. The volume of the permeates collected from the filtration was measured. A total of nine trials (Table 2) with different processing conditions were formed according to the orthogonal design.

| Determination of total polyphenol content
The total polyphenol content of the permeate after filtration was determined in accordance with the Folin-Ciocalteu method described by Kajal et al. (2015) with minor modifications. An aliquot of 1 ml of each permeates after filtration prepared from each of the nine trials was mixed with 5 ml of the Folin-Ciocalteu reagent (10% in distilled water) in a test tube. After 5 min, 4 ml of sodium carbonate (7.5% in distilled water) was added to each tube before the test tubes were cap screwed and vortexed. The samples were incubated for 2 h at room temperature in darkness. The absorbance was measured at 725 nm with a UV-vis spectrophotometer (Ultrospec 3000 pro, Amersham Pharmacia Biotech, Ltd.). A standard curve with serial phloroglucinol solutions (ranging from 20 lg/ml to 100 lg/ml) was used for calibration. The total polyphenol content in the permeate after filtration was calculated as the polyphenol content of 1 ml aliquot times the volume ratio of permeate after filtration (Table 3).

| Antioxidation measurement
Three measurements, DPPH radical scavenging activity, hydroxyl radical scavenging activity, and lipid peroxidation, were utilized to investigate antioxidative activity. The three permeates after filtration produced from optimized conditions were freeze dried to powder, then redissolved in 70% ethanol at a ratio of 10% (w/v). The redissolved solutions were used for antioxidation measurements.

| DPPH radical scavenging activity measurement
The DPPH radical scavenging activity method was utilized to inves- (1)

| Gas chromatography-mass spectrometry (GC-MS) analysis
The polyphenol complexes extracted from the three seaweed species under the optimized processing condition of each were subjected to gas chromatography-mass spectrometry (GC-MS) analyses (Rahaman et al., 2019(Rahaman et al., , 2020. GC-MS analyses were performed on a Thermo Fisher (San Jose, CA) TRACE DSQ single-quadrupole mass spectrometer following the method developed by Verslues (2017) with slight modifications. The GC conditions were as follows: column, were as follows: ionization, electron impact (70 eV); detection, positive ion; full-scan analyses, 10 m/z-600 m/z at 2 scans/s. Volatile metabolites were eluted with the solvent front using this method, so GC separation of these analyses started with an initial temperature of 40°C held for 2 min, followed by an increase to 80°C at 10°C/min.
The temperature was maintained for 3 min at 80°C after which it was increased to 230°C at a rate of 30°C/min.

| Data analysis
Measurements were performed in triplicate. Data were presented as the mean with standard deviation and subjected to one-way analysis of variance (ANOVA) and least significant difference (LSD) using MINITAB Statistical Software v15. The significance was judged statistically by the F value at probability (p) below .05.  The higher K value of each column indicated stronger impact. This demonstrated that the optimized processing conditions for the different seaweed species of Sargassum miyabei, Undaria pinnatifida suringar, and Sargassum thunbergii were A 2 B 1 C 3 D 1 (processing temperature of 60°C, ethanol concentration of 70%, ratio of powder to liquid of 1:10, and processing time of 3 h); A 3 B 1 C 1 D 2 (processing temperature of 70°C, ethanol concentration of 70%, ratio of powder to liquid of 1:8, and processing time of 4 h), and A 3 B 1 C 1 D 2 (processing temperature of 7°C, ethanol concentration of 70%, ratio of powder to liquid of 1:8, and processing time of 4 h), respectively. The total polyphenol content produced from the optimized conditions of each seaweed species was compared with that from the nine trials. It was found that for each seaweed species, the total polyphenol content of the permeate after filtration produced by optimized processing conditions significantly exceeded from each of the nine trials of the same species. Among others, the total polyphenol content produced by the optimized processing conditions from Sargassum thunbergii was significantly higher than the others.

| Antioxidative activity of polyphenols produced from optimized processing conditions of Sargassum miyabei, Undaria Pinnatifida Suringar, and Sargassum thunbergii
The antioxidative activity of hydrolysates has been reported in polyphenols extracted from different food sources such as almond (Bolling, 2017), black tea (Tenore et al., 2015), and algae (Machu et al., 2015). Our study found that among the polyphenols extracted against anthocyanin degradation and color loss in vitamin C fortified cranberry juice. They concluded that gallic acid was able to preserve health beneficial components and the endogenous red color in cranberry juice after 16 days of storage time. However, the control sample without gallic acid did not show these characteristics. Fatemeh et al. (2015) found that the antioxidant capacity of 50 mg/kg/bw arbutin had a protective effect on lipid peroxidation and cyclosporine-induced toxicity. Furthermore, Erenler et al. (2016) reported that the compounds extracted from Origanum majorana exhibited significant antioxidant activities.
They stated that the arbutin was one of the major compounds in the sample. Furthermore, DPPH free radical scavenging assay proved that the antioxidative activity of arbutin (45%) was the highest followed by butylated hydroxytoluene (15%). However, the antioxidative activity of syringate ( Figure 1a) and phloretin ( Figure 1c) has rarely been reported.

CO N FLI C T O F I NTE R E S T
All authors declare no conflicting interests.